Microorganisms for producing glycogen and methods of using same

ABSTRACT

Recombinant microorganisms configured for increased glycogen production. The recombinant microorganisms comprise a recombinant nucleic acid configured to express or overexpress a glucose-1-phosphate adenylyltransferase. The recombinant microorganisms produce an increased amount of glycogen compared to a corresponding microorganism not comprising the recombinant nucleic acid.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under GE01215871 andEFRI1240268 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The invention is directed to recombinant microorganisms configured forproducing high levels of glycogen and methods of using the recombinantmicroorganisms for the production of glycogen or its byproducts.

BACKGROUND

Advances in microbe engineering for the production of biofuels,chemicals, and therapeutics have spurred investment in the production ofa wide variety of commodities from biological sources (Zhang F,Rodriguez S, Keasling J D. 2011. Curr. Opin. Biotechnol. 22(6):775-83).Heterotrophic microbes comprise the vast majority of microorganismscurrently utilized for product generation and require a carbohydratesource for carbon and energy that can account for a significantproportion (˜60%) of input costs (Pimentel D, Patzek T W. 2008. Ethanolproduction: energy and economic issues related to U.S. and Braziliansugarcane biofuels. Springer, Amsterdam, Netherlands.). Suchcarbohydrate feedstocks are typically derived from agricultural crops,primarily sugarcane, sugar beet, and corn, although lignocellulosicmaterials are under extensive investigation as alternative feedstocks(Sims R, Taylor M. 2008. From 1st to 2nd generation biofueltechnologies. IEA, Paris, France). While biologically produced fuels andchemicals hold the promise of increased sustainability and reduced CO₂footprints, current feedstock sources place biotechnological processesin competition with agricultural croplands and food markets. Thedevelopment of biological alternatives to standard petroleum-based fuelsand chemicals has therefore been criticized for its capacity to increasefood cost and instabilities (Timilsina G R, Beghin J C, van derMensbrugghe D, Mevel S. 2010. The impacts of biofuel targets on land-usechange and food supply. The World Bank Development Research Group,Washington, D.C.). Indeed, in recent years, sugar prices have increasedand fluctuated greatly in global food, driven in part by increaseddemands for biofuel production.

Photosynthetic microorganisms (cyanobacteria and algae) have beenproposed as alternative sources for the creation of biofuel-likecompounds or industrial feedstocks (Radakovits R, Jinkerson R E, DarzinsA, Posewitz M C. 2010. Eukaryot. Cell 9:486-501), in part because theypossess many advantages over traditional terrestrial plants with regardto targeted metabolite production. For example, the photosyntheticefficiency of cyanobacteria is up to an order of magnitude higher thanthat of plants (Zhu X G, Long S P, Ort D R. 2010. Annu. Rev. Plant Biol.61:235-261) (Zhu X G, Long S P, Ort D R. 2008. Curr. Opin. Biotechnol.19:153-159), and cyanobacteria do not require support tissues thatfurther reduce productive output (e.g., roots/stems). Cyanobacteria aregenetically tractable, allowing for rapid engineering and the selectionof desirable strains. Finally, cyanobacteria are aquatic microbes withminimal nutritional requirements and can therefore be cultivated inlocations that do not compete with traditional agricultural crops. Whilecyanobacteria and algae share many similar features in this context, theuse of algal species for biofuel feedstocks has been explored in muchgreater detail, partly because of their relatively high lipid content(Sheehan J, Dunahay T, Benemann J, Roessler P. 1998. Look back at theU.S. Department of Energy's aquatic species program: biodiesel fromalgae. Close-out report NREL/TP-580-24190. National Renewable EnergyLaboratory, Golden, Colo.), although many cyanobacterial species featurerelative simplicity and higher growth rates.

Glycogen that accumulates in microorganisms can serve as a valuablefeedstock for the production of chemicals and biofuels. Glycogen can beconverted to ethanol or other chemicals, for example, throughsaccharification and fermentation processes (Aikawa et al. Energ EnvironSci 2013, 6:1844-1849) (Choi et al. Bioresour Technol 2010,101:5330-5336) (Harun et al. Appl Energy 2011, 88:3464-3467) (Ho et al.Bioresour Technol 2013, 145:142-149) (Miranda et al. Bioresour Technol2012, 104:342-348).

There is a need for microorganisms capable of producing high amounts ofglycogen or other carbohydrates, particularly through photosyntheticprocesses.

SUMMARY OF THE INVENTION

The present invention is directed at least in part to microorganisms,such as photosynthetic microorganisms, that are capable of producinghigh levels of glycogen; methods of producing glycogen; and methods forselecting microorganisms that produce high levels of glycogen or othermetabolic products.

The objects and advantages of the invention will appear more fully fromthe following detailed description of the preferred embodiment of theinvention made in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show glycogen production in control strains and strains ofthe invention in the presence of 0, 0.5 or 5 mM isopropylβ-D-1-thiogalactopyranoside (IPTG).

FIG. 2 shows results of a glycogen production screen of the inventionwith control strains and strains of the invention induced to produceglycogen in the presence of ambient CO₂ and 0 or 0.5 mM (IPTG).

FIG. 3 shows results of a glycogen production screen of the inventionwith control strains and strains of the invention induced to produceglycogen in the presence of 10% CO₂ and 0, 0.1, or 0.5 mM (IPTG).

FIG. 4 shows intracellular levels of glycogen as hydrolyzed glucose fromthe strains analyzed in FIG. 3.

FIG. 5 shows growth rates of a control strain and a strain of theinvention grown in the presence of 0 mM or 0.1 mM IPTG.

FIGS. 6, 7A, and 7B show levels of glucose hydrolyzed from glycogen froma control strain and a strain of the invention grown in the presence of0 mM or 0.1 mM IPTG for various lengths of time.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed at least in part to microorganisms capable ofenhanced production of glycogen.

The microorganism of the present invention may include any microorganismcapable of making glycogen. The microorganism may be eukaryotic, such asyeast, or prokaryotic, such as bacteria or archaea. Among bacteria,gram-positive, gram-negative, and ungrouped bacteria are suitable.Phototrophs, chemotrophs, heterotrophs, and autotrophs (e.g.,chemoautotrophs, photoautotrophs, chemoheterotrophs, photoheterotrophs)are suitable. The phototroph may be an anoxygenic photosyntheticmicroorganism or an oxygenic photosynthetic mircoorganism. The oxygenicphotosynthetic microorganism may be a cyanobacterium or a microalga.Suitable cyanobacteria include those from the genuses Agmenellum,Anabaena, Aphanocapsa, Arthrosprira, Gloeocapsa, Haplosiphon,Mastigocladus, Nostoc, Oscillatoria, Prochlorococcus, Scytonema,Synechococcus, and Synechocystis. Preferred cyanobacteria include thoseselected from the group consisting of Synechococcus spp., spp.,Synechocystis spp., and Nostoc spp. Particularly suitable examples ofSynechococcus spp. include Synechococcus sp. PCC 7942 and Synechococcussp. PCC 7002. A particularly suitable example of Synechocystis spp.includes Synechocystis sp. PCC 6803. A benefit of photoautotrophs suchas cyanobacteria is that they require only CO₂ as a carbon source andlight for energy and are not dependent on food-based commodities orother types of biomass for which there is a growing high demand.

The microorganisms of the invention may be modified to increaseexpression of one or more enzymes. Modifying the microorganism toincrease expression of an enzyme can be performed using any methodscurrently known in the art or discovered in the future. Examples includegenetically modifying the microorganism and culturing the microorganismunder conditions or in the presence of factors that increase expressionof the enzyme. Suitable methods for genetic modification include but arenot limited to placing the coding sequence under the control of a moreactive promoter (either inducible or constitutive), increasing gene copynumber, introducing a translational enhancer (see, e.g., Olins et al.Journal of Biological Chemistry, 1989, 264(29):16973-16976), and/orincreasing expression of transactivators. Increasing gene copy numbercan be performed by introducing additional copies of the gene to themicroorganism, i.e., by incorporating one or more exogenous copies ofthe native gene or a heterologous homolog thereof into the microbialgenome, by introducing such copies to the microorganism on a plasmid orother vector, or by other means. “Exogenous” used in reference to agenetic element means the genetic element is either not present in thenative organism or is not present in the native organism in the sameconfiguration. “Heterologous” used in reference to a genetic elementmeans that the genetic element is derived from a different species. Apromoter that controls a particular coding sequence is herein describedas being “operationally connected” to the coding sequence.

The microorganisms of the invention may include at least one recombinantnucleic acid configured to express or overexpress a particular enzyme.“Recombinant” as used herein with reference to a nucleic acid moleculeor polypeptide is one that has a sequence that is not naturallyoccurring, such as a sequence that made by an artificial combination oftwo otherwise separated segments of sequence from the same or differentorganisms, or a sequence made by artificial combination of a naturallyoccurring sequence with a non-naturally occurring sequence. Thisartificial combination can be achieved, for example, by chemicalsynthesis or by the artificial manipulation of isolated segments ofnucleic acid molecules or polypeptides, such as genetic engineeringtechniques. “Recombinant” is also used to describe nucleic acidmolecules that have been artificially modified but contain the sameregulatory sequences and coding regions that are found in the organismfrom which the nucleic acid was isolated. A recombinant cell ormicroorganism is one that contains a recombinant nucleic acid moleculeor polypeptide. “Overexpress” as used herein means that a particulargene product is produced at a higher level in one cell, such as arecombinant cell, than in a corresponding microorganism. For example, amicroorganism that includes a recombinant nucleic acid configured tooverexpress an enzyme produces the enzyme at a greater amount than amicroorganism that does not include the recombinant nucleic acid.

As used herein, “corresponding microorganism” refers to a microorganismof the same species having the same or substantially same genetic andproteomic composition as a microorganism of the invention, with theexception of genetic and proteomic differences resulting from themodifications described herein for the microorganisms of the invention.In some versions, the corresponding microorganism is the nativemicroorganism. “Native” in this context refers to the natural,unmodified microorganism as it exists in nature.

Some microorganisms of the invention include at least one recombinantnucleic acid configured to express or overexpress a glucose-1-phosphateadenylyltransferase. The recombinant nucleic acid may comprise arecombinant glucose-1-phosphate adenylyltransferase gene. “Gene” refersto a nucleic acid sequence capable of producing a gene product and mayinclude such genetic elements as a coding sequence together with anyother genetic elements required for transcription and/or translation ofthe coding sequence. Such genetic elements may include a promoter, anenhancer, and/or a ribosome binding site (RBS), among others. Therecombinant gene preferably comprises at least one sequence differencefrom the natural gene.

Glucose-1-phosphate adenylyltransferase include enzymes classified underEC 2.7.7.27. Glucose-1-phosphate adenylyltransferase include enzymesthat catalyze the conversion of adenosine triphosphate (ATP) andα-D-glucose 1-phosphate to diphosphate and adenosine diphosphate(ADP)-glucose. In some versions, the microorganism is modified to harbora nucleic acid encoding a glucose-1-phosphate adenylyltransferase fromEscherichia coli or a homolog thereof. An exemplary coding sequence fora glucose-1-phosphate adenylyltransferase (glgC) from E. coli isrepresented by SEQ ID NO: 1. An exemplary amino acid sequence for aglucose-1-phosphate adenylyltransferase from E. coli (GlgC) isrepresented by SEQ ID NO:2. The native glucose-1-phosphateadenylyltransferase from E. coli has been shown to be activated byfructose-1,6-bisphosphate and inhibited by adenosine monophosphate (AMP)and ADP through allosteric regulation.

Homologs of the E. coli glucose-1-phosphate adenylyltransferase includeorthologs and paralogs of GlgC/glgC having glucose-1-phosphateadenylyltransferase activity. Homologs of the E. coliglucose-1-phosphate adenylyltransferase also include enzymes having anamino acid sequence at least about 80%, 85%, 90%, 95%, 97%, 98%, 99% ormore identical to SEQ ID NO:2. Sequences having these percent identitiescan be obtained by aligning SEQ ID NO:2 to the sequences of E. coliglucose-1-phosphate adenylyltransferase orthologs and/or paralogs havingglucose-1-phosphate adenylyltransferase activity to determine whichpositions in the enzyme are amenable to mutation (i.e., substitution,deletion, addition, etc.) and the identities of the substituted or addedresidues at these positions.

In preferred versions of the invention, the glucose-1-phosphateadenylyltransferase expressed by the microorganism maintains allostericregulation by AMP and/or ADP. In particularly preferred versions of theinvention, the glucose-1-phosphate adenylyltransferase expressed by themicroorganism maintains full allosteric regulation by AMP and/or ADP.The maintenance of allosteric regulation with the glucose-1-phosphateadenylyltransferase is determined with respect to the wild-typeglucose-1-phosphate adenylyltransferase in the type of organism fromwhich the glucose-1-phosphate adenylyltransferase is derived, wherein“wild-type” refers to the allele that encodes the phenotype most commonin the natural population. Variants or “mutants” of glucose-1-phosphateadenylyltransferase resistant to allosteric regulation by AMP and ADPare known. See, e.g., Leung P, Lee Y M, Greenberg E, Esch K, Boylan S,Preiss J. Cloning and expression of the Escherichia coli glgC gene froma mutant containing an ADPglucose pyrophosphorylase with alteredallosteric properties. J Bacterial. 1986 July; 167(1):82-8. One suchvariant is the E. coli GlgC variant having a G336D substitution (codingsequence: SEQ ID NO:3; protein sequence: SEQ ID NO:4). The G336D varianthas reduced allosteric regulation with respect to the wild-type E. coliglgC represented by SEQ ID NO:2 and is a more active form of the enzyme.Expression of the G336D variant in cyanobacteria, however, adverselyaffects growth rate. Expression of glucose-1-phosphateadenylyltransferases that have a glycine at a position corresponding toposition 336 of SEQ ID NO:2 (E. coli GlgC) are therefore preferred. Insome versions, however, expression of glucose-1-phosphateadenylyltransferases that have an amino acid other than glycine at aposition corresponding to position 336 of SEQ ID NO:2 (E. coli GlgC) areacceptable. Exemplary amino acids other than glycine include acidicamino acids, such as glutamic acid and aspartic acid, among others.Identification of the corresponding position in a given sequence can befound by aligning the sequence with SEQ ID NO:2.

The glucose-1-phosphate adenylyltransferase expressed by themicroorganism preferably maintains allosteric regulation by AMP and/orADP to an extent such that 50% inhibition of the glucose-1-phosphateadenylyltransferase occurs at an AMP or ADP concentration+/−about10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold,1.5-fold, or 1.1-fold of the AMP or ADP concentration that induces 50%inhibition of the wild-type glucose-1-phosphate adenylyltransferase.

In some versions of the invention, the glucose-1-phosphateadenylyltransferase expressed by the microorganism maintains allostericregulation by AMP, ADP, and/or fructose-1,6-bisphosphate.

Exogenous, heterologous nucleic acids encoding enzymes to be expressedin the microorganism are preferably codon-optimized for the particularmicroorganism in which they are introduced. Codon optimization can beperformed for any nucleic acid by a number of programs, including“GENEGPS”-brand expression optimization algorithm by DNA 2.0 (MenloPark, Calif.), “GENEOPTIMIZER”-brand gene optimization software by LifeTechnologies (Grand Island, N.Y.), and “OPTIMUMGENE”-brand gene designsystem by GenScript (Piscataway, N.J.). Other codon optimizationprograms or services are well known and commercially available.

In some versions of the invention, the microorganism exhibits a nativeglycogen synthase expression level. “Native glycogen synthase expressionlevel” refers to the level of glycogen synthase expression in thenative, unmodified microorganism. In such versions, the microorganism isnot modified to overexpress the glycogen synthase, whereinoverexpression is defined with respect to expression in the nativemicroorganism. Examples of a glycogen synthase in bacteria such as E.coli and cyanobacteria include products of glgA genes. Examples ofproducts of glgA genes include glgA1 (SEQ ID NO:6) and glgA2 (SEQ IDNO:8) of Synechococcus sp. PCC 7002, encoded by glgA1 (SEQ ID NO:5) andglgA2 (SEQ ID NO:7), respectively. Accordingly, in at least someversions of the invention in which the microorganism exhibits a nativeglycogen synthase expression level the microorganism contains the nativeglgA gene(s) and/or does not include a recombinant glgA gene configuredto overexpress glgA.

In some versions of the invention, the microorganism exhibits nativeglycogen synthase activity. “Native glycogen synthase activity” refersto the level of glycogen synthase activity in the native, unmodifiedmicroorganism. Glycogen synthase activity in the microorganism may bedetermined by the method described by Leung et al. (Leung P, Lee Y M,Greenberg E, Esch K, Boylan S, Preiss J. Cloning and expression of theEscherichia coli glgC gene from a mutant containing an ADPglucosepyrophosphorylase with altered allosteric properties. J Bacteriol. 1986July; 167(1):82-8) and Kawajuchi et al. (Kawaguchi K, Fox J, Holmes E,Boyer C, Preiss J. De novo synthesis of Escherichia coli glycogen is dueto primer associated with glycogen synthase and activation by branchingenzyme. Arch Biochem Biophys. 1978 October; 190(2):385-97).

In some versions of the invention, the microorganism exhibits a native1,4-alpha-glucan-branching enzyme expression level. “Native1,4-alpha-glucan-branching enzyme expression level expression level”refers to the level of 1,4-alpha-glucan-branching enzyme expression inthe native, unmodified microorganism. In such versions, themicroorganism is not modified to overexpress the1,4-alpha-glucan-branching enzyme, wherein overexpression is definedwith respect to expression in the native (non-modified) microorganism.Examples of a 1,4-alpha-glucan-branching enzyme in bacteria such as E.coli and cyanobacteria include products of glgB genes. An example of aproduct of a glgB gene includes glgB (SEQ ID NO:10) of Synechococcus sp.PCC 7002, which is encoded by glgB (SEQ ID NO:9). Accordingly, in atleast some versions of the invention in which the microorganism exhibitsa native 1,4-alpha-glucan-branching enzyme expression level themicroorganism contains the native glgB gene(s) and/or does not include arecombinant glgB gene configured to overexpress glgB.

In some versions of the invention, the microorganism exhibits native1,4-alpha-glucan-branching enzyme activity. “Native1,4-alpha-glucan-branching enzyme activity” refers to the level of1,4-alpha-glucan-branching enzyme activity in the native, unmodifiedmicroorganism. 1,4-Alpha-glucan-branching enzyme activity in themicroorganism may be determined by the method described by Leung et al.(Leung P, Lee Y M, Greenberg E, Esch K, Boylan S, Preiss J. Cloning andexpression of the Escherichia coli glgC gene from a mutant containing anADPglucose pyrophosphorylase with altered allosteric properties. JBacteriol. 1986 July; 167(1):82-8) and Boyer et al. (Boyer C, Preiss J.Biosynthesis of bacterial glycogen. Purification and properties of theEscherichia coli B alpha-1,4,-glucan: alpha-1,4-glucan6-glycosyltansferase. Biochemistry. 1977 Aug. 9; 16(16):3693-9.).

In some versions of the invention, the microorganism exhibits a nativefructose-bisphosphate aldolase enzyme expression level. “Nativefructose-bisphosphate aldolase enzyme expression level expression level”refers to the level of fructose-bisphosphate aldolase enzyme expressionin the native, unmodified microorganism. In such versions, themicroorganism is not modified to overexpress the fructose-bisphosphatealdolase enzyme, wherein overexpression is defined with respect toexpression in the native (non-modified) microorganism. Examples of afructose-bisphosphate aldolase enzyme in bacteria such as E. coli andcyanobacteria include products of fba genes. An example of a product ofa fba gene includes fba (SEQ ID NO:12) of Synechocystis sp. PCC 6803,which is encoded by fba (SEQ ID NO:11). Accordingly, in at least someversions of the invention in which the microorganism exhibits a nativefructose-bisphosphate aldolase enzyme expression level the microorganismcontains the native fba gene(s) and/or does not include a recombinantfba gene configured to overexpress fba. In some versions of theinvention, the microorganism exhibits native fructose-bisphosphatealdolase enzyme activity.

In some versions of the invention, the microorganism exhibits a nativefructose 1,6-bisphosphatase enzyme expression level. “Native fructose1,6-bisphosphatase enzyme expression level expression level” refers tothe level of fructose 1,6-bisphosphatase enzyme expression in thenative, unmodified microorganism. In such versions, the microorganism isnot modified to overexpress the fructose 1,6-bisphosphatase enzyme,wherein overexpression is defined with respect to expression in thenative (non-modified) microorganism. Examples of a fructose1,6-bisphosphatase enzyme in bacteria such as E. coli and cyanobacteriainclude products of fbp genes. An example of a product of a fbp geneincludes fbp (SEQ ID NO:14) of Synechocystis sp. PCC 6803, which isencoded by fbp (SEQ ID NO:11). Accordingly, in at least some versions ofthe invention in which the microorganism exhibits a native fructose1,6-bisphosphatase enzyme expression level the microorganism containsthe native fbp gene(s) and/or does not include a recombinant fba geneconfigured to overexpress fbp. In some versions of the invention, themicroorganism exhibits native fructose 1,6-bisphosphatase enzymeactivity.

The microorganism of the invention may comprise modifications thatreduce or ablate the activity of gene products of one or more genes.Such a modification that that reduces or ablates the activity of geneproducts of one or more genes is referred to herein as a “functionaldeletion” of the gene product. “Gene product” refers to a protein orpolypeptide encoded and produced by a particular gene.

One of ordinary skill in the art will appreciate that there are manywell-known ways to functionally delete a gene product. For example,functional deletion can be accomplished by introducing one or moregenetic modifications. As used herein, “genetic modifications” refer toany differences in the nucleic acid composition of a cell, whether inthe cell's native chromosome or in endogenous or exogenousnon-chromosomal plasmids harbored within the cell. Examples of geneticmodifications that may result in a functionally deleted gene productinclude but are not limited to mutations such as substitutions, partialor complete deletions, insertions, or other variations to a codingsequence or a sequence controlling the transcription or translation of acoding sequence; placing a coding sequence under the control of a lessactive promoter; blocking transcription of the gene with a trans-actingDNA binding protein such as a TAL effector or CRISPR guided Cas9;expressing ribozymes or antisense sequences that target the mRNA of thegene of interest; and tagging proteins for rapid proteolytic decay(Cameron D E, Collins J J. Tunable protein degradation in bacteria. NatBiotechnol. 2014 December; 32(12):1276-81.), etc. In some versions, agene or coding sequence can be replaced with a selection marker orscreenable marker. Various methods for introducing the geneticmodifications described above are well known in the art and includehomologous recombination, among other mechanisms. See, e.g., Green etal., Molecular Cloning: A laboratory manual, 4^(th) ed., Cold SpringHarbor Laboratory Press (2012) and Sambrook et al., Molecular Cloning: ALaboratory Manual, 3^(rd) ed., Cold Spring Harbor Laboratory Press(2001). Various other genetic modifications that functionally delete agene product are described in the examples below. Functional deletioncan also be accomplished by inhibiting the activity of the gene product,for example, by chemically inhibiting a gene product with a smallmolecule inhibitor, by expressing a protein that interferes with theactivity of the gene product, or by other means.

In certain versions of the invention, the functionally deleted geneproduct may have less than about 95%, less than about 90%, less thanabout 85%, less than about 80%, less than about 75%, less than about70%, less than about 65%, less than about 60%, less than about 55%, lessthan about 50%, less than about 45%, less than about 40%, less thanabout 35%, less than about 30%, less than about 25%, less than about20%, less than about 15%, less than about 10%, less than about 5%, lessthan about 1%, or about 0% of the activity of the non-functionallydeleted gene product.

In certain versions of the invention, a cell with a functionally deletedgene product may have less than about 95%, less than about 90%, lessthan about 85%, less than about 80%, less than about 75%, less thanabout 70%, less than about 65%, less than about 60%, less than about55%, less than about 50%, less than about 45%, less than about 40%, lessthan about 35%, less than about 30%, less than about 25%, less thanabout 20%, less than about 15%, less than about 10%, less than about 5%,less than about 1%, or about 0% of the activity of the gene productcompared to a cell with the non-functionally deleted gene product.

In certain versions of the invention, the functionally deleted geneproduct may be expressed at an amount less than about 95%, less thanabout 90%, less than about 85%, less than about 80%, less than about75%, less than about 70%, less than about 65%, less than about 60%, lessthan about 55%, less than about 50%, less than about 45%, less thanabout 40%, less than about 35%, less than about 30%, less than about25%, less than about 20%, less than about 15%, less than about 10%, lessthan about 5%, less than about 1%, or about 0% of the amount of thenon-functionally deleted gene product.

In certain versions of the invention, the functionally deleted geneproduct may result from a genetic modification in which at least 1, atleast 2, at least 3, at least 4, at least 5, at least 10, at least 20,at least 30, at least 40, at least 50, or more nonsynonymoussubstitutions are present in the gene or coding sequence of the geneproduct.

In certain versions of the invention, the functionally deleted geneproduct may result from a genetic modification in which at least 1, atleast 2, at least 3, at least 4, at least 5, at least 10, at least 20,at least 30, at least 40, at least 50, or more bases are inserted in thegene or coding sequence of the gene product.

In certain versions of the invention, the functionally deleted geneproduct may result from a genetic modification in which at least about1%, at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or about 100% of the gene product's gene or coding sequenceis deleted or mutated.

In certain versions of the invention, the functionally deleted geneproduct may result from a genetic modification in which at least about1%, at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or about 100% of a promoter driving expression of the geneproduct is deleted or mutated.

In certain versions of the invention, the functionally deleted geneproduct may result from a genetic modification in which at least about1%, at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or about 100% of an enhancer controlling transcription of thegene product's gene is deleted or mutated.

In certain versions of the invention, the functionally deleted geneproduct may result from a genetic modification in which at least about1%, at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or about 100% of a sequence controlling translation of geneproduct's mRNA is deleted or mutated.

In certain versions of the invention, the decreased activity orexpression of the functionally deleted gene product is determined withrespect to the activity or expression of the gene product in itsunaltered state as found in nature. In certain versions of theinvention, the decreased activity or expression of the functionallydeleted gene product is determined with respect to the activity orexpression of the gene product in its form in a correspondingmicroorganism. In certain versions, the genetic modifications givingrise to a functionally deleted gene product are determined with respectto the gene or coding sequence in its unaltered state as found innature. In certain versions, the genetic modifications giving rise to afunctionally deleted gene product are determined with respect to thegene or coding sequence in its form in a corresponding microorganism.

Homologs include genes or gene products (including enzymes) that arederived, naturally or artificially, from a common ancestral gene or geneproduct. Homology is generally inferred from sequence similarity betweentwo or more genes or gene products. Homology between genes may beinferred from sequence similarity between the products of the genes. Theprecise percentage of similarity between sequences that is useful inestablishing homology varies with the gene or gene product at issue, butas little as 25% sequence similarity (e.g., identity) over 50, 100, 150or more residues (nucleotides or amino acids) is routinely used toestablish homology (e.g., over the full length of the two sequences tobe compared). Higher levels of sequence similarity (e.g., identity),e.g., 30%, 35% 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 99% or more, can also be used to establish homology.Accordingly, homologs of the coding sequences, genes, or gene productsdescribed herein include coding sequences, genes, or gene products,respectively, having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the codingsequences, genes, or gene products, respectively, described herein. Insome versions, homologs of the genes described herein include genes thathave gene products at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the gene productsof the genes described herein. Methods for determining sequencesimilarity percentages (e.g., BLASTP and BLASTN using defaultparameters) are described herein and are generally available. Thehomologous gene products should demonstrate comparable activities and,if an enzyme, participate in the same or analogous pathways. “Orthologs”are genes or coding sequences thereof in different species that evolvedfrom a common ancestral gene by speciation. Normally, orthologs retainthe same or similar function in the course of evolution. As used herein“orthologs” are included in the term “homologs.” Homologs also includeparalogs.

For sequence comparison and homology determination, one sequencetypically acts as a reference sequence to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence based on the designated program parameters. A typicalreference sequence of the invention is a nucleic acid or amino acidsequence corresponding to coding sequences, genes, or gene productsdescribed herein.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (supplemented through 2008)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity for purposes of defininghomologs is the BLAST algorithm, which is described in Altschul et al.,J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysesis publicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always>0)and N (penalty score for mismatching residues; always<0). For amino acidsequences, a scoring matrix is used to calculate the cumulative score.Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001. The above-describedtechniques are useful in identifying homologous sequences for use in themethods described herein.

The terms “identical” or “percent identity”, in the context of two ormore nucleic acid or polypeptide sequences, refers to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms described above (or otheralgorithms available to persons of skill) or by visual inspection.

The phrase “substantially identical”, in the context of two nucleicacids or polypeptides refers to two or more sequences or subsequencesthat have at least about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90, about 95%, about 98%, or about 99% or morenucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using a sequence comparisonalgorithm or by visual inspection. Such “substantially identical”sequences are typically considered to be “homologous” without referenceto actual ancestry. Preferably, the “substantial identity” exists over aregion of the sequences that is at least about 50 residues in length,more preferably over a region of at least about 100 residues, and mostpreferably, the sequences are substantially identical over at leastabout 150 residues, at least about 250 residues, or over the full lengthof the two sequences to be compared.

Accordingly, homologs of the genes described herein include genes withgene products at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or moreidentical to the gene products of the genes described herein.

In some versions, the microorganisms of the invention produce anincreased amount of glycogen compared to a corresponding microorganismnot comprising the modifications described herein. For example, themicroorganisms of the invention may be capable of producing at leastabout 1.1-fold, about 1.25-fold, about 1.5-fold, about 1.75-fold, about2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3-foldor more glycogen than a corresponding microorganism, and/or up to about2.5-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, ormore glycogen than a corresponding microorganism.

In some versions, the microorganisms of the invention produce glycogenat an increased rate compared to a corresponding microorganism notcomprising the modifications described herein. For example, themicroorganisms of the invention may be capable of producing glycogen ata rate at least about 2-fold, about 3-fold, about 4-fold, about 5-fold,about 6-fold, about 7-fold, about 8-fold, about 9-fold or more than acorresponding microorganism, and/or up to about 5-fold, about 10-fold,about 12-fold, about 15-fold or more than a corresponding microorganism.

In some versions, the microorganisms of the invention produce glycogenat a rate of at least about 50 mg/L/day, about 100 mg/L/day, about 125mg/L/day, about 150 mg/L/day, about 175 mg/L/day, about 200 mg/L/day, ormore, and/or up to about 190 mg/L/day, about 200 mg/L/day, about 225mg/L/day, about 250 mg/L/day, about 275 mg/L/day, about 300 mg/L/day ormore.

In some versions, the microorganisms of the invention are capable ofproducing glycogen as a mass percent of dry cell weight (DCW) in anamount of at least about 10% DCW, at least about 15% DCW, at least about20% DCW, at least about 25% DCW, at least about 26% DCW, at least about27% DCW, at least about 28% DCW, at least about 29% DCW, at least about30% DCW, at least about 31% DCW, at least about 32% DCW, at least about33% DCW, at least about 34% DCW, or at about least 35% DCW and/or up toabout or at least about 31% DCW, about or at least about 32% DCW, aboutor at least about 33% DCW, about or at least about 34% DCW, about or atleast about 35% DCW, about or at least about 36% DCW, about or at leastabout 37% DCW, about or at least about 38% DCW, about or at least about39% DCW, or about or at least about 40% DCW.

In some versions, the microorganisms of the invention have a growth ratesubstantially the same as a corresponding microorganism when culturedunder identical conditions, such that the modifications described hereindo not substantially affect the growth rate. For example, themicroorganisms of the invention may have a growth rate within about 40%,about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about5%, about 3%, about 2%, or about 1% the growth rate of a correspondingmicroorganism when cultured under identical conditions. In someversions, the microorganisms of the invention have a growth rate of atleast the growth rate of a corresponding microorganism when culturedunder identical conditions.

In addition to the microorganism itself, the invention also providesmethods of producing glycogen with the microorganisms of the presentinvention. The methods involve culturing the microorganism in conditionssuitable for growth of the microorganism. Such conditions includeproviding suitable carbon and energy sources for the particularmicroorganism. Suitable carbon and energy sources for particular typesof microorganisms are described elsewhere herein for exemplarymicroorganisms and are otherwise known in the art.

The invention also provides methods of screening for production ofglycogen or other metabolic products. The screening methods generallyinvolve culturing microorganisms under conditions that promoteproduction of the metabolic product, then stressing the microorganismsunder stringent conditions that promote consumption of the metabolicproduct at a high metabolic rate, and then comparing the recovery ratesof the microorganisms when reintroduced to more suitable growthconditions.

An exemplary screening method includes culturing microorganisms in thepresence of a carbon source and a first amount of an energy source underconditions suitable for producing the metabolic product, then culturingthe microorganisms in the presence of a second amount of the energysource under conditions suitable for consuming the metabolic product,then culturing the microorganisms in the presence of the carbon sourceand a third amount of the energy source and determining the relativegrowth of the microorganisms in the presence of the carbon source andthe third amount of the energy source. The second amount of the energysource is preferably less than the first amount of the energy source,and the third amount of the energy source is preferably greater than thesecond amount of the energy source.

The metabolic product preferably comprises a product comprising reducedcarbon that serves as a form of stored energy for the microorganism andis consumable by the microorganism for survival when a sufficientexternal energy source is lacking. Such products may includecarbohydrates, lipids, and/or proteins. Exemplary carbohydrates mayinclude simple carbohydrates such as monosaccharides or disaccharides orcomplex carbohydrates such as trisaccharides, tetrasaccharides, starch,or glycogen, among others. Exemplary lipids may include fatty acids,glycerol, or glycerides, among others.

The energy source may comprise a fermentable or oxidizable form ofreduced molecules, if the microorganism is a chemotroph, or light, ifthe microorganism is an autotroph. The reduced molecules may be organicor inorganic. Examples of reduced organic molecules include reducedcarbon, such as carbohydrates, lipids, proteins, methane, and otherreduced organic molecules. Reduced organic molecules can be used forchemoorganotrophs. Examples of reduced inorganic molecules includeiron(II), Mn²⁺, H₂, sulfide (H₂S), inorganic sulfur (S₀), thiosulfate(S₂O₃ ²⁻), ammonia, and nitrite, among others. Reduced inorganicmolecules can be used for chemolithotrophs.

The carbon source may comprise organic carbon, if the microorganism is ahetrotroph, or carbon dioxide, if the microorganism is an autotroph.Examples of organic carbon include carbohydrates, lipids, and proteins.

The microorganisms used in the selection method may comprise anymicroorganism described herein.

The conditions suitable for consuming the metabolic product preferablycomprise a temperature sufficient to support metabolic activity of themicroorganisms in the presence of the second amount of the energysource. Such a temperature may be at least about 27° C., at least about30° C., at least about 35° C., at least about 37° C., at least about 40°C. or more and/or up to about 37° C., up to about 40° C., up to about45° C. or more.

In exemplary versions of the invention, the microorganisms comprisephotosynthetic microorganisms, the carbon source comprises CO₂, theenergy source comprises light, and the metabolic product comprisesglycogen. Culturing the microorganisms in the first amount of the energysource may comprise exposing the microorganisms to a direct source oflight. Culturing the microorganisms in the second amount of the energysource may comprise substantially blocking the microorganisms from anydirect source of light. Culturing the microorganisms in the third amountof the energy source may comprise exposing the microorganisms to adirect source of light. The photosynthetic microorganisms may comprisecyanobacteria and/or microalgae.

The elements and method steps described herein can be used in anycombination whether explicitly described or not.

All combinations of method steps as used herein can be performed in anyorder, unless otherwise specified or clearly implied to the contrary bythe context in which the referenced combination is made.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the content clearly dictates otherwise.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, from 5to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All patents, patent publications, and peer-reviewed publications (i.e.,“references”) cited herein are expressly incorporated by reference tothe same extent as if each individual reference were specifically andindividually indicated as being incorporated by reference. In case ofconflict between the present disclosure and the incorporated references,the present disclosure controls.

It is understood that the invention is not confined to the particularconstruction and arrangement of parts herein illustrated and described,but embraces such modified forms thereof as come within the scope of theclaims.

Examples

Strains

fba (coding sequence: SEQ ID NO:11; protein sequence: SEQ ID NO:12) andfbp (coding sequence: SEQ ID NO:13; protein sequence: SEQ ID NO:14) fromSynechocystis PCC 6803 was inserted as an operon into the cLac143 IPTGinducible cassette described in Markley et al. 2015 (Markley A L,Begemann M B, Clarke R E, Gordon G C, Pfleger B F. ACS Synth Biol. 2015May 15; 4(5):595-603) with 500 base pair flanking regions targeting theacsA locus in PCC 7002, forming construct pALM173 (SEQ ID NO:15). WildType glgC from K12 MG1655 E. coli genomic DNA (coding sequence: SEQ IDNO:1; protein sequence: SEQ ID NO:2) was inserted into the cLac94 IPTGinducible cassette described in Markley et al. 2015 with 500 base-pairflanking regions targeting the glpK locus in PCC 7002, forming constructpALM210 (SEQ ID NO:16). glgC with a G336D mutation (coding sequence: SEQID NO:3; protein sequence: SEQ ID NO:4) was amplified from a BioBrickpart BBa K118016 and inserted into an identical vector backbone aspALM210 to form pALM211 (SEQ ID NO:17).

These genetic elements were inserted into the PCC 7002 chromosome byadding 1-1.5 μg of purified plasmid DNA to 1 mL of an overnight cultureof cells grown to an OD₇₃₀ of 1. The cultures were then placed at 37° C.under illumination for 16 hours. The cells were plated on 50 μM acrylicacid (acsA locus) or 100 μg/ml gentamycin (glpK locus) to select forrecombinants. This yielded strains were AM184 (WT 7002 AcsA::cLac143FbaFbp), AM241 (WT 7002 glpK::cLac94 GlgC K12 GmR) and AM253 (WT 7002glpK::cLac94 GlgC K12 G336D GmR). Double fba-fbp/glgC strains wereconstructed by repeating the pALM210/pALM211 glgC transformations in theAM184 fba-fbp strain to produce AM254 (AM184 7002 glpK::cLac94 GlgC K12GmR) and AM255 (AM184 7002 glpK::cLac94 GlgC K12 G336D GmR).

The generated strains are shown in Table 1.

TABLE 1 Strains used in the present examples. Strain Parent Construct IDDescription Strain Name(s) AM184 WT 7002 AcsA::cLac143 FbaFbp PCC 7002pALM173 Fix AM241 WT 7002 glpK::cLac94 GlgC K12 PCC 7002 pALM210 GmRAM253 WT 7002 glpK::cLac94 GlgC K12 PCC 7002 pALM211 G336D GmR AM254AM184 7002 glpK::cLac94 GlgC PCC 7002 pALM173 + K12 GmR pALM210 AM255AM184 7002 glpK::cLac94 GlgC PCC 7002 pALM173 + K12 G336D GmR pALM211Initial Glycogen Production Testing

Initial experiments on the produced strains were performed in CorningCostar non-treated 6-well tissue culture plates with 6 mL of MediaA⁺(0.308 M NaCl, 0.02 M MgSO₄.7H₂O, 0.08 mM Na₂EDTA.2H₂O, 8.05 mM KCl,2.52 mM CaCl₂.2H₂O, 11.8 mM NaNO₃, 0.37 mM KH₂PO₄, 8.26 mM TRIZMA® base(Sigma-Aldrich, St. Louis, Mo.) pH 8.2, 55.5 mM H₃BO₃, 0.23 mM ZnCl₂,0.021 mM MoO₃(85%), 0.3 μM vitamin B12 (cyanocobalamin), 0.14 mMFeCl₃.6H₂O, 0.22 mM MnCl₂.4H₂O, 0.00012 mM CuSO₄.5H₂O, 0.0005 mMCoCl₂.6H₂O) according to the UTEX Culture Collection of Algae at TheUniversity of Texas at Austin. IPTG was added at 0.5 mM and 5 mM. Thecultures were grown on a shaker at 37° C. under illumination for 2 days.The induced strains with glgC G336D (AM253 and AM255) had a severegrowth defect. 1.5 OD₇₃₀*mL were collected and pelleted. The pelletswere washed 3× with PBS and analyzed for glycogen content using theGlycogen Assay Kit (Item No. 700480) from Cayman Chemical Company (AnnArbor, Mich.).

To prepare samples for the glycogen assay with the Glycogen Assay Kit,1× Glycogen Assay Buffer was prepared according to the manufacturer'sinstructions. 1.5 OD ml (approximately 400 mg DCW based on my standardcurve) was taken from each culture in regular Media A at low CO₂ andwashed 3× in PBS to remove Tris interference. Cell pellets wereresuspended in 2 ml Diluted Assay Buffer+1×PMSF. Samples were frozen at−80° C. until further use. To finish the sample preparation for theglycogen assay, the remaining reagents were prepared according to themanufacturer's instructions. The frozen samples were sonicated on ice at20% amplitude in 2-second bursts for 1 min total. The sample preparationwas finished according to the manufacturer's instructions while alsotesting different dilution factors. The assay was then performedaccording to the manufacturer's instructions.

AM253 (glgC G336D) and AM255 (glgC G336D+fba−fbp) yielded inconsistentglycogen yields with these experiments, likely due to their poor growthrates. Additionally, while strains containing the glgC G336D had a highglycogen:dry cell weight ratio, the low growth rate resulted in a loweroverall productivity when compared with WT glgC strains. See FIGS.1A-1C.

Since these experiments showed that AM241 and AM254 had 2-3 fold moreglycogen than WT PCC 7002 without a severe growth defect, these strainswere chosen for further testing.

Glycogen Production Screen

In order to aid in the testing of the glycogen-producing strains, ascreen was developed that couples glycogen content to cellular fitness.The overall scheme of this screen is to grow strains in liquid mediausing any desired growth condition. The cells are then normalized to thesame OD₇₃₀ and serially diluted in sterile MediaA⁺. 7.5 μl of thesedilutions are then spotted on several replicate MediaA⁺ agar plates. Oneplate is immediately placed under illumination at 37° C. while theremaining plates are placed in a dark 37° C. incubator. The plates arethen periodically removed from the dark incubator and placed in thelight. Cells that have a high glycogen content show higher recoveryrates compared to cells with low or no glycogen content.

An alternative strategy whereby liquid cultures of high and low glycogencontent strains were incubated in the dark at 37° C. for several daysand periodically spotted on MediaA⁺ agar plates before outgrowing in theilluminated growth chamber showed no difference in cellular fitnessbetween strains. Similarly, simply leaving the solid agar plates at roomtemperature instead of 37° C. also did not work as well due to the veryslow loss in fitness.

Testing of Strains Using the Glycogen Production Screen

The glycogen production screen described above was performed on WT PCC7002, a native glgC knockout (through kanamycin resistance geneinactivation), AM241, and AM254. Each strain was inoculated at 0.05OD₇₃₀ in 20 mL of MediaA⁺ and grown for 16 hours at 37° C. in thepresence of ambient CO₂ and 0 or 0.5 mM IPTG. After 16 hours, thestrains were normalized to the same OD₇₃₀, serially diluted, spotted onMediaA⁺ agar plates, placed in a dark 37° C. incubator for variousamounts of time, and placed in the light to determine relative recovery.Results are shown in FIG. 2.

The glycogen production screen was also performed on the same strainsgrown in 10% CO₂. For this experiment, WT PCC 7002, the native glgCknockout, AM241, and AM254 were inoculated at 0.05 OD₇₃₀ in 20 mL ofMediaA⁺ and grown for 16 hours at 37° C. with 10% CO₂ by volume bubbledinto the tubes in the presence of 0, 0.1, or 0.5 mM IPTG. After 16hours, the strains were normalized to the same OD₇₃₀, serially diluted,spotted on MediaA⁺ agar plates, placed in a dark 37° C. incubator forvarious amounts of time, and placed in the light to determine relativerecovery. Results are shown in FIG. 3. Additionally, after the 16-hoursof growth, 10 OD₇₃₀*mL of each sample were spun down and lyophilizedthen resuspended with 1 mL of 4% H₂SO₄ and placed at 121° C. for onehour to hydrolyze the glycogen to glucose. After an hour, the sampleswere neutralized up to a pH of >2 and then run on an HPLC with a Bio-radAminex HPX-87H Sugar Byproducts Column using a 5 mM H₂SO₄ isocraticrunning buffer. The glucose peaks were compared with a standard curve todetermine intracellular sugar content. Results are shown in FIG. 4. Thesugar content of the cells was highly correlative to the relativesurvival rate in the dark. Compare FIGS. 4 and 3, respectively.

The AM241 (glpK::cLac94 glgC K12 WT) strain was chosen for larger scalebioreactor studies.

Bioreactor Runs

Approximately 250 mL of WT PCC 7002 and AM241 bacteria were grown underambient CO₂ conditions in the light, and then these cultures were usedto inoculate 900 mL MediaA⁺ bioreactors in triplicate at an OD₇₃₀ of0.01. Six total bioreactors of AM241 were inoculated, and IPTG was addedto three of them to a final concentration of 0.1 mM IPTG. Thebioreactors were then grown at 37° C. with 10% CO₂, and 60 OD₇₃₀*mL werecollected periodically and analyzed for sugar content by HPLC asdescribed above. There was no significant difference in growth ratesbetween the WT and AM241 cultures (FIG. 5), but AM241 induced at 0.1 mMIPTG showed a 3.2 fold increase in glycogen content over WT 7002 and atiter of 476 mg/L glycogen after 64 hours (FIG. 6). Critically, this isdone without having to lower the growth rate of the cyanobacteria ormodify the nutrient ratios, as has been the only strategy for glycogenproduction in cyanobacteria.

Total glycogen content did start to decrease after 64 hours. See FIGS.7A and 7B. This decrease was likely due to IPTG degradation. It ispredicted that use of a constitutive expression system will prevent sucha decrease.

Additional parameters from the bioreactor experiments are shown in Table2.

TABLE 2 Sample parameters of bioreactor runs. Dry Cell DCW/ HPLC PercentGlucose Days of Weight Sample Sample Glucose Glucose/ Glucose TotalProduction Growth (DCW) Volume Volume Content Sample of Glucose Rate(Days) Strain (mg) (mL) (mg/L) (mg/ml) (mg) DCW (mg/L) (mg/L/day) 1.1 WT7002 27.3 41.78 653.40 0.29 0.62  2.3% 14.83 14 24.8 34.40 720.90 0.390.83  3.4% 24.24 22 24.2 38.36 630.80 0.39 0.83  3.4% 21.67 20 AM24123.8 42.02 566.40 1.75 3.76 15.8% 89.41 83   0 mM 16.8 37.41 449.10 2.004.30 25.6% 115.02 106 22.6 43.35 521.30 1.82 3.92 17.4% 90.49 84 AM24124.2 39.37 614.70 3.66 7.87 32.5% 199.86 184 0.1 mM 26.1 38.76 673.403.82 8.22 31.5% 212.01 196 23.5 37.69 623.50 3.44 7.39 31.4% 196.03 1812.7 WT 7002 17.2 12.76 1358.80 0.60 1.30  7.5% 102.38 38 24.4 11.672090.30 1.08 2.33  9.6% 199.77 75 25.0 13.57 1841.70 0.87 1.88  7.5%138.32 52 AM241 24.5 13.89 1764.00 1.21 2.61 10.6% 187.58 70   0 mM 24.912.77 1950.50 1.64 3.52 14.1% 275.91 103 24.1 14.08 1711.10 1.39 2.9912.4% 212.50 80 AM241 25.3 13.10 1931.20 2.84 6.11 24.1% 466.33 175 0.1mM 24.3 12.82 1895.40 2.83 6.09 25.1% 474.95 178 25.8 13.57 1900.60 3.086.62 25.6% 487.45 183 3.6 WT 7002 25.7 9.12 2818.40 1.36 2.92 11.4%320.66 88 32.9 7.71 4266.00 2.60 5.60 17.0% 726.09 200 28.8 9.71 2966.40AM241 23.6 8.04 2934.30 1.37 2.94 12.4% 365.24 137   0 mM 26.0 8.722981.30 2.23 4.79 18.4% 549.06 206 27.3 10.34 2639.00 AM241 33.5 9.843405.80 2.68 5.77 17.2% 586.13 220 0.1 mM 32.0 9.74 3285.30 2.72 5.8418.3% 599.88 225 64.7 9.68 6685.70 2.71 5.83  9.0% 602.30 226 4.6 WT7002 27.2 7.71 3526.90 2.82 6.06 22.3% 785.14 170 25.6 6.67 3840.00 3.868.30 32.4% 1244.80 269 25.1 7.21 3480.50 2.79 6.00 23.9% 832.15 180AM241 26.8 7.61 3519.70 3.09 6.65 24.8% 873.35 189   0 mM 26.0 7.193614.00 3.62 7.79 30.0% 1082.64 234 26.5 8.26 3206.50 3.06 6.57 24.8%795.44 172 AM241 27.9 7.13 3915.30 3.25 6.98 25.0% 979.12 212 0.1 mM25.4 7.03 3615.30 3.41 7.34 28.9% 1044.75 226 29.0 7.46 3886.00 3.006.44 22.2% 863.37 187 5.6 WT 7002 25.5 6.06 4207.50 3.57 7.68 30.1%1267.58 225 24.6 5.69 4316.70 3.97 8.54 34.8% 1502.08 267 24.1 6.253856.00 3.57 7.68 31.9% 1229.12 219 AM241 24.8 6.22 3988.70 3.76 8.0932.6% 1301.43 231   0 mM 23.0 5.97 3852.50 4.07 8.75 38.1% 1466.38 26124.3 6.67 3645.00 3.66 7.86 32.4% 1179.50 210 AM241 28.6 6.19 4623.704.62 9.93 34.7% 1604.57 285 0.1 mM 25.1 5.94 4225.20 4.25 9.15 36.4%1539.74 274 27.0 6.03 4477.50 3.84 8.25 30.6% 1368.33 243 6.7 WT 700225.6 5.60 4573.90 3.73 8.02 31.3% 1432.83 215 25.9 5.70 4541.10 4.7510.20 39.4% 1788.97 268 25.6 6.00 4266.70 3.45 7.41 28.9% 1234.81 185AM241 24.7 5.62 4396.60 3.23 6.95 28.1% 1236.35 185   0 mM 25.4 6.074182.50 4.24 9.11 35.9% 1499.46 225 25.2 6.05 4166.40 3.61 7.76 30.8%1283.22 192 AM241 28.5 5.58 5111.00 4.38 9.42 33.0% 1689.06 253 0.1 mM26.1 5.47 4767.60 3.92 8.42 32.3% 1538.89 231 28.8 5.70 5049.60 3.647.82 27.1% 1370.34 206

What is claimed is:
 1. A recombinant microorganism modified with respectto a native microorganism, the recombinant microorganism comprising arecombinant nucleic acid configured to express a glucose-1-phosphateadenylyltransferase, wherein the recombinant microorganism: is acyanobacterium; exhibits enhanced glucose-1-phosphateadenylyltransferase activity compared to the native microorganism;exhibits a native glycogen synthase expression level; exhibits nativeglycogen synthase activity; and produces an increased amount of glycogencompared to the native microorganism while having a growth rate of atleast a growth rate of the native microorganism when grownphotoautotrophically in the presence of light and 10% CO₂.
 2. Therecombinant microorganism of claim 1, wherein the glucose-1-phosphateadenylyltransferase is allosterically regulated by a compound selectedfrom the group consisting of adenosine diphosphate and adenosinemonophosphate.
 3. The recombinant microorganism of claim 1, wherein theglucose-1-phosphate adenylyltransferase comprises a sequence at least90% identical to SEQ ID NO:2.
 4. The recombinant microorganism of claim1, wherein the glucose-1-phosphate adenylyltransferase comprises aglycine at a position corresponding to position 336 of SEQ ID NO:2. 5.The recombinant microorganism of claim 1, wherein the nucleic acidcomprises a glucose-1-phosphate adenylyltransferase coding sequenceoperably connected to a promoter not operably connected to the codingsequence in nature.
 6. The recombinant microorganism of claim 5, whereinthe promoter is an inducible promoter.
 7. The recombinant microorganismof claim 5, wherein the promoter is a constitutive promoter.
 8. Therecombinant microorganism of claim 1, wherein the recombinantmicroorganism exhibits a native 1,4-alpha-glucan-branching enzymeexpression level.
 9. The recombinant microorganism of claim 1, whereinthe recombinant microorganism exhibits native 1,4-alpha-glucan-branchingenzyme activity.
 10. The recombinant microorganism of claim 1, whereinthe recombinant microorganism is capable of producing glycogen as a masspercent of dry cell weight (DCW) in an amount of at least about 25% DCW.11. A method of producing glycogen comprising culturing the recombinantmicroorganism of claim
 1. 12. The recombinant microorganism of claim 1,wherein the recombinant microorganism exhibits a native1,4-alpha-glucan-branching enzyme expression level and native1,4-alpha-glucan-branching enzyme activity.
 13. The recombinantmicroorganism of claim 12, wherein the recombinant microorganism iscapable of producing glycogen as a mass percent of dry cell weight (DCW)in an amount of at least about 25% DCW.
 14. The recombinantmicroorganism of claim 13, wherein the glucose-1-phosphateadenylyltransferase is allosterically regulated by a compound selectedfrom the group consisting of adenosine diphosphate and adenosinemonophosphate.
 15. The recombinant microorganism of claim 13, whereinthe glucose-1-phosphate adenylyltransferase comprises a sequence atleast 90% identical to SEQ ID NO:2.
 16. The recombinant microorganism ofclaim 13, wherein the glucose-1-phosphate adenylyltransferase comprisesa glycine at a position corresponding to position 336 of SEQ ID NO:2.17. The recombinant microorganism of claim 13, wherein the nucleic acidcomprises a glucose-1-phosphate adenylyltransferase coding sequenceoperably connected to a promoter not operably connected to the codingsequence in nature.
 18. The recombinant microorganism of claim 17,wherein the promoter is an inducible promoter.
 19. The recombinantmicroorganism of claim 17, wherein the promoter is a constitutivepromoter.
 20. A method of producing glycogen comprising culturing therecombinant microorganism of claim 13.